Poliovirus and related human enteroviruses cause a diverse spectrum of human diseases including acute flaccid paralysis (paralytic poliomyelitis), aseptic meningitis, myocarditis, conjunctivitis and fatal systemic infections of neonates. Poliovirus (PV), the prototypic human enterovirus, is well characterized at the molecular level and serves as a model organism for detailed studies of human enterovirus mRNA translation, RNA replication, and virus assembly. In this proposal we will exploit a remarkable cell-free replication system to investigate how the mechanisms of PV mRNA stability, PV mRNA translation, PV RNA replication, and PV assembly work in concert to evade host cell mRNA turnover machinery and a dsRNA-activated antiviral pathway. In the previous funding period we made three important discoveries: 1) we discovered mechanisms by which naturally uncapped PV mRNA assembles polysomes uncoupled from host cell mRNA turnover machinery, 2) we discovered an evolutionarily ancient telomerase-like mechanism of RNA replication that maintains the integrity of the poly(A) tail at the 3'end of viral RNA genomes, and 3) we discovered an RNA structure in PV and related group C enteroviruses that functions as a competitive inhibitor of ribonuclease L, an antiviral endoribonuclease. Experiments will be performed to: 1) determine how PV and related human enteroviruses uncouple viral mRNA translation from host cell mRNA turnover pathways, 2) elucidate molecular features associated with the telomerase-like mechanism of RNA replication, and 3) test whether the viral RNA structure that competitively inhibits ribonuclease L works best within the confines of RNA replication complexes. These investigations will reveal how the seemingly arcane mechanisms of viral mRNA translation, viral RNA replication, and virus assembly represent concerted strategies shaped by the selective pressures of host mRNA turnover machinery and dsRNA-activated antiviral host responses. These investigations will provide a clear and sophisticated understanding of the manner in which metabolic steps of viral replication are directly influenced by host RNA degradation pathways. These insights will be important for designing safe and effective vaccines for PV and other group C enteroviruses as well as antiviral drug therapies for many positive-strand RNA viruses.